Methods for predicting the survival time of patients with decompensated alcoholic cirrhosis

ABSTRACT

The present invention relates to methods for predicting the survival time of patients with decompensated alcoholic cirrhosis. In particular, the present invention relates to a method for predicting the survival time of a patient with decompensated alcoholic cirrhosis comprising i) determining the expression level of OAS2 or MX2 in a sample of peripheral blood mononuclear cells obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value and iii) and concluding that the patient will have a short survival time when the level determined at step i) is higher than its predetermined reference value or concluding that the patient will have a long survival time when the level determined at step i) is lower than the predetermined reference value.

FIELD OF THE INVENTION

The present invention relates to methods for predicting the survivaltime of patients with decompensated alcoholic cirrhosis.

BACKGROUND OF THE INVENTION

Cirrhosis is a chronic disease of the liver whose prevalence willdramatically increase during the next decade. Cirrhosis can result froma number of chronic liver diseases such as alcoholic liver disease,chronic viral hepatitis, non-alcoholic steatohepatitis, autoimmunediseases of the liver (primary biliary cirrhosis, primary sclerosingcholangitis, and autoimmune hepatitis). Cirrhosis progresses overseveral years. The occurrence of complications indicates the transitionto the phase called “decompensated” (approximately 100,000 patients peryear in France); These complications include ascites (30 000 patientsper year in France), gastrointestinal bleeding (10,000 episodes/year inFrance), renal failure and bacterial infections which is very common andoften due to the translocation of Gram-negative intestinal bacteria.Mortality in cirrhosis is thus usually a consequence of decompensationor its ensuing complications. The treatment of choice for decompensatedcirrhosis is liver transplantation and many such patients are placed ontransplant waiting lists. Therefore predicting the survival time ofpatients with decompensated alcoholic cirrhosis is highly desirable fordetermining whether the patient shall be eligible to transplantation.The MELD (Model for End Stage Liver Disease) score is currently used fororgan allocation. Although the MELD score predicts 90-day mortalitybased on bilirubin, INR (international normalized ratio) and serumcreatinine, the predisposing factors for death and final events leadingto mortality need to be improved.

SUMMARY OF THE INVENTION

The present invention relates to methods for predicting the survivaltime of patients with decompensated alcoholic cirrhosis. In particular,the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Although systemic inflammation is believed to be a major driver ofmortality in patients with decompensated alcoholic cirrhosis, itsmechanisms are unclear. On the basis of the results of microarray geneexpression profiling performed in cirrhotic and healthy immune cells,the inventors hypothesized that baseline expression levels of genesinvolved in cell-autonomous immunity (most of which are type 1IFN-inducible) and/or of genes encoding secreted inflammatory cytokinesor chemokines by circulating mononuclear cells in patients withcirrhosis may be related to patient outcome. Thus, they measured thebaseline gene expression in peripheral blood mononuclear cells (PBMCs)from patients with decompensated alcoholic cirrhosis and investigatedtheir relationship with the risk of death. The inventors demonstratethat in PBMCs from patients with decompensated alcoholic cirrhosis,higher baseline levels of type 1 IFN-inducible genes involved incell-autonomous immunity, but not expression of genes encoding secretedinflammatory cytokines/chemokines, are highly predictive of the risk ofdeath. Deregulation of type 1 IFN-inducible gene expression incirculating immune cells may play a role in the mechanisms resulting indeath from cirrhosis. More particularly, in univariate analysis, ahigher MELD score, higher baseline expression of 8 IFN-inducible genesas well as higher IFN score values were significant predictors of death.In bivariate analysis including the IFN and MELD scores, only the formersignificantly predicted death (RR=3.58; 95% CI, 1.18-10.91; P=0.02).These results were mainly due to the elevated intrinsic prognostic valueof OAS2 (RR=2.49; P=0.04) and MX2 (RR=1.35; P=0.01).

Accordingly a first object of the present invention relates to a methodfor predicting the survival time of a patient with decompensatedalcoholic cirrhosis comprising i) determining the expression level ofOAS2 or MX2 in a sample of peripheral blood mononuclear cells obtainedfrom the patient, ii) comparing the level determined at step i) with apredetermined reference value and iii) and concluding that the patientwill have a short survival time when the level determined at step i) ishigher than its predetermined reference value or concluding that thepatient will have a long survival time when the level determined at stepi) is lower than the predetermined reference value.

As used herein the expression “decompensated alcoholic cirrhosis” hasits general meaning in the art. In cirrhosis, the presence of jaundice,ascites, portal hypertensive gastrointestinal bleeding, and/or, hepaticencephalopathy, or any combination of these is considereddecompensation. The above manifestations appear when the disease processoverwhelms the compensatory mechanisms, either by disease progression ora superimposed acute insult.

The method is particularly suitable for predicting the duration of theoverall survival (OS) of the patient. Those of skill in the art willrecognize that OS survival time is generally based on and expressed asthe percentage of people who survive for a specific amount of time. Asused herein, the expression “short survival time” indicates that thepatient will have a survival time that will be lower than the median (ormean) observed in the general population of patient with decompensatedalcoholic cirrhosis. When the patient will have a short survival time,it is meant that the patient will have a “poor prognosis” and is at highrisk of death on liver transplant waiting list. Inversely, theexpression “long survival time” indicates that the patient will have asurvival time that will be higher than the median (or mean) observed inthe general population of patient with decompensated alcoholic cirrhosisand that he may survive until liver transplantation. When the patientwill have a long survival time, it is meant that the patient will have a“good prognosis”.

The term “PBMC” or “peripheral blood mononuclear cells” or“unfractionated PBMC”, as used herein, refers to whole PBMC, i.e. to apopulation of white blood cells having a round nucleus, which has notbeen enriched for a given sub-population. Cord blood mononuclear cellsare further included in this definition. Typically, the PBMC sampleaccording to the invention has not been subjected to a selection step tocontain only adherent PBMC (which consist essentially of >90% monocytes)or non-adherent PBMC (which contain T cells, B cells, natural killer(NK) cells, NK T cells and DC precursors). A PBMC sample according tothe invention therefore contains lymphocytes (B cells, T cells, NKcells, NKT cells), monocytes, and precursors thereof. Typically, thesecells can be extracted from whole blood using Ficoll, a hydrophilicpolysaccharide that separates layers of blood, with the PBMC forming acell ring under a layer of plasma. Additionally, PBMC can be extractedfrom whole blood using a hypotonic lysis buffer which willpreferentially lyse red blood cells. Such procedures are known to theexpert in the art.

In some embodiments, both the expression levels of OAS2 and MX2 aredetermined at step i).

In some embodiments, the expression level of at least one further geneis determined and compared to its corresponding predetermined value. Insome embodiments, the gene for which the expression is furtherdetermined is selected from the group consisting of IFIT1, CXCL10,IFIH1, DDX58, TRIM22 and GBP4.

In some embodiments, the expression levels of 2, 3, 4, 5, 6, 7 or 8genes are determined.

In some embodiments, the expression levels of OAS2, MX2, IFIT1, CXCL10,IFIH1, DDX58, TRIM22 and GBP4 are determined in the sample and comparedto their corresponding predetermined reference value.

In a further aspect, the expression level of at least one gene selectedfrom the group consisting of OAS2, MX2, IFIT1, CXCL10, IFIH1, DDX58,TRIM22, GBP4, DDX60, IFIT5, IFI44 and IRF1 are determined in the sampleand compared to their corresponding predetermined reference value

In some embodiments, the expression levels of 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 genes are determined.

In the present specification, the name of each of the genes of interestrefers to the official gene symbol of the corresponding gene, as foundin internationally recognised gene sequences and protein sequencesdatabases, in particular in the database from the HUGO Gene NomenclatureCommittee, that is available notably at the following Internet address:www.gene.ucl.ac.uk/nomenclature/index.html. In the presentspecification, the name of each of the various biological markers ofinterest may also refer to the internationally recognised name of thecorresponding gene, as found in the internationally recognised genesequences and protein sequences databases ENTRE ID, Genbank, TrEMBL orENSEMBL. Through these internationally recognised sequence databases,the nucleic acid sequences corresponding to each of the gene of interestdescribed herein may be retrieved by the one skilled in the art. Foravoidance of doubt, each gene of the present invention is characterizedin Table A with its access number available onwww.ncbi.nlm.nih.gov/gene/:

TABLE A Genes included in the IFN Score with corresponding locus,encoded protein and function Gene Location Protein Function Entrez IDDDX58 9p21.1 RIG-I RNA Helicase, PRR for viral RNA 23586 OAS2 12q24.132′-5′-oligoadenylate 2-5A synthetase responsible for RNase L 4939synthetase 2 activation and degradation of viral RNA MX2 21q22.3Interferon-induced GTP- Anti-viral activity (HIV and SIV). May 4600binding protein Mx2 play a role in regulating nucleocytoplasmictransport and cell-cycle progression TRIM22 11p15.4 Tripartitemotif-containing 22 Interferon-induced antiviral protein involved 10346in cell innate immunity. The antiviral activity could in part bemediated by TRIM22- dependent ubiquitination of viral proteins GBP41p22.2 Guanylate binding protein 4 Binds GTP, GDP and GMP. HydrolyzesGTP with 115361 GDP or GMP being reaction products CXCL10 4q21.1 C-X-Cmotif chemokine 10 Chemotactic for monocytes and T-lymphocytes. 3627Binds to CXCR3. IFIT1 10q23.31 IFN-induced protein Antiviral RNA-bindingprotein acting as a with tetratricopeptide sensor of viralsingle-stranded RNAs and repeats 1 inhibiting expression of viral mRNA.IFIH1 2q24.2 Melanoma Differentiation- RIG-I-like receptor dsRNAhelicase enzyme 64135 Associated protein 5 acting as a cytoplasmicsensor of viral RNA. Major role in sensing viral infection and inactivation of antiviral responses.

TABLE B Further genes included in the method of the invention withcorresponding encoded protein and function Gene Protein Function EntrezID DDX60 DEXD/ Positively regulates DDX58/RIG-I- 55601 H-box andIFIH1/MDA5-dependent type I helicase interferon and interferon 60inducible gene expression in response to viral infection. Binds ssRNA,dsRNA and dsDNA and can promote the binding of DDX58/RIG-I to dsRNA.IFIT5 Interferon Interferon-induced RNA-binding 24138 induced proteinthat specifically binds protein with single-stranded RNA bearing atetratrico- 5′-triphosphate group (PPP- peptide RNA), thereby acting asa sensor repeats 5 of viral single-stranded RNAs. Single-strandedPPP-RNAs are specific from viruses, providing a molecular signature todistin- guish between self and non-self mRNAs. IFI44 Interferon Thisprotein aggregates to form 10561 induced microtubular structures protein44 IRF1 Interferon Serves as an activator of inter- 3659 regulatoryferons alpha and beta transcrip- factor 1 tion (required fordouble-stranded RNA induction of these genes in mouse). Also functionsas a tran- scription activator of genes in- duced by interferons alpha,beta, and gamma. Plays also roles in regulating apoptosis and tumor-suppression

Determination of the expression level of a gene can be performed by avariety of techniques. Generally, the expression level as determined isa relative expression level. Typically, the determination comprisescontacting the sample with selective reagents such as probes, primers orligands, and thereby detecting the presence, or measuring the amount, ofpolypeptide or nucleic acids of interest originally in the sample.Contacting may be performed in any suitable device, such as a plate,microtiter dish, test tube, well, glass, column, and so forth. In someembodiments, the contacting is performed on a substrate coated with thereagent, such as a nucleic acid array or a specific ligand array. Thesubstrate may be a solid or semi-solid substrate such as any suitablesupport comprising glass, plastic, nylon, paper, metal, polymers and thelike. The substrate may be of various forms and sizes, such as a slide,a membrane, a bead, a column, a gel, etc. The contacting may be madeunder any condition suitable for a detectable complex, such as a nucleicacid hybrid or an antibody-antigen complex, to be formed between thereagent and the nucleic acids or polypeptides of the sample.

In some embodiments, the expression level may be determined bydetermining the quantity of mRNA. Methods for determining the quantityof mRNA are well known in the art. For example the nucleic acidcontained in the sample is first extracted according to standardmethods, for example using lytic enzymes or chemical solutions orextracted by nucleic-acid-binding resins following the manufacturer'sinstructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR).Typically quantitative or semi-quantitative RT-PCR is preferred.Real-time quantitative or semi-quantitative RT-PCR is particularlyadvantageous. Other methods of Amplification include ligase chainreaction (LCR), transcription-mediated amplification (TMA), stranddisplacement amplification (SDA) and nucleic acid sequence basedamplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequencecomplementarity or homology to the mRNA of interest herein find utilityas hybridization probes or amplification primers. It is understood thatsuch nucleic acids need not be identical, but are typically at leastabout 80% identical to the homologous region of comparable size, morepreferably 85% identical and even more preferably 90-95% identical. Incertain embodiments, it will be advantageous to use nucleic acids incombination with appropriate means, such as a detectable label, fordetecting hybridization.

Typically, the nucleic acid probes include one or more labels, forexample to permit detection of a target nucleic acid molecule using thedisclosed probes. In various applications, such as in situ hybridizationprocedures, a nucleic acid probe includes a label (e.g., a detectablelabel). A “detectable label” is a molecule or material that can be usedto produce a detectable signal that indicates the presence orconcentration of the probe (particularly the bound or hybridized probe)in a sample. Thus, a labeled nucleic acid molecule provides an indicatorof the presence or concentration of a target nucleic acid sequence(e.g., genomic target nucleic acid sequence) (to which the labeleduniquely specific nucleic acid molecule is bound or hybridized) in asample. A label associated with one or more nucleic acid molecules (suchas a probe generated by the disclosed methods) can be detected eitherdirectly or indirectly. A label can be detected by any known or yet tobe discovered mechanism including absorption, emission and/or scatteringof a photon (including radio frequency, microwave frequency, infraredfrequency, visible frequency and ultra-violet frequency photons).Detectable labels include colored, fluorescent, phosphorescent andluminescent molecules and materials, catalysts (such as enzymes) thatconvert one substance into another substance to provide a detectabledifference (such as by converting a colorless substance into a coloredsubstance or vice versa, or by producing a precipitate or increasingsample turbidity), haptens that can be detected by antibody bindinginteractions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules(or fluorochromes). Numerous fluorochromes are known to those of skillin the art, and can be selected, for example from Life Technologies(formerly Invitrogen), e.g., see, The Handbook—A Guide to FluorescentProbes and Labeling Technologies). Examples of particular fluorophoresthat can be attached (for example, chemically conjugated) to a nucleicacid molecule (such as a uniquely specific binding region) are providedin U.S. Pat. No. 5,866,366 to Nazarenko et al., such as4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl) amino naphthalene-1-sulfonic acid (EDANS),4-amino-N-[³ vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, antl1ranilamide,Brilliant Yellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); cyanosine;4′,6-diarninidino-2-phenylindole (DAPI);5′,5″dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulforlic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6diclllorotriazin-2-yDarninofluorescein (DTAF),2′7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC Q(RITC);2′,7′-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B,sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine101 (Texas Red); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA);tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);riboflavin; rosolic acid and terbium chelate derivatives. Other suitablefluorophores include thiol-reactive europium chelates which emit atapproximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27,1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, Lissamine™,diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein,4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No.5,800,996 to Lee et al.) and derivatives thereof. Other fluorophoresknown to those skilled in the art can also be used, for example thoseavailable from Life Technologies (Invitrogen; Molecular Probes (Eugene,Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, asdescribed in U.S. Pat. Nos. 5,696,157, 6,130,101 and 6,716,979), theBODIPY series of dyes (dipyrrometheneboron difluoride dyes, for exampleas described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782,5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an aminereactive derivative of the sulfonated pyrene described in U.S. Pat. No.5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent labelcan be a fluorescent nanoparticle, such as a semiconductor nanocrystal,e.g., a QUANTUM DOT™ (obtained, for example, from Life Technologies(QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.);see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).Semiconductor nanocrystals are microscopic particles havingsize-dependent optical and/or electrical properties. When semiconductornanocrystals are illuminated with a primary energy source, a secondaryemission of energy occurs of a frequency that corresponds to the handgapof the semiconductor material used in the semiconductor nanocrystal.This emission can he detected as colored light of a specific wavelengthor fluorescence. Semiconductor nanocrystals with different spectralcharacteristics are described in e.g., U.S. Pat. No. 6,602,671.Semiconductor nanocrystals that can he coupled to a variety ofbiological molecules (including dNTPs and/or nucleic acids) orsubstrates by techniques described in, for example, Bruchez et al.,Science 281:20132016, 1998; Chan et al., Science 281:2016-2018, 1998;and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals ofvarious compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927,069;6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736;6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807;5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Puhlication No.2003/0165951 as well as PCT Puhlication No. 99/26299 (puhlished May 27,1999). Separate populations of semiconductor nanocrystals can heproduced that are identifiable based on their different spectralcharacteristics. For example, semiconductor nanocrystals can he producedthat emit light of different colors hased on their composition, size orsize and composition. For example, quantum dots that emit light atdifferent wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mnemission wavelengths), which are suitable as fluorescent labels in theprobes disclosed herein are available from Life Technologies (Carlshad,Calif.).

Quantitative PCR is typically carried out in a thermal cycler with thecapacity to illuminate each sample with a beam of light of a specifiedwavelength and detect the fluorescence emitted by the excitedfluorophore. The thermal cycler is also able to rapidly heat and chillsamples, thereby taking advantage of the physicochemical properties ofthe nucleic acids and thermal polymerase. The majority of thethermocyclers on the market now offer similar characteristics.Typically, thermocyclers involve a format of glass capillaries, plasticstubes, 96-well plates or 384-wells plates. The thermocylcer also involvea software analysis. In order to detect and measure the amount ofamplicon (i.e. amplified target nucleic acid sequence) in the sample, ameasurable signal has to be generated, which is proportional to theamount of amplified product.

In some embodiments, the expression level is determined by DNA chipanalysis. Such DNA chip or nucleic acid microarray consists of differentnucleic acid probes that are chemically attached to a substrate, whichcan be a microchip, a glass slide or a microsphere-sized bead. Amicrochip may be constituted of polymers, plastics, resins,polysaccharides, silica or silica-based materials, carbon, metals,inorganic glasses, or nitrocellulose. Probes comprise nucleic acids suchas cDNAs or oligonucleotides that may be about 10 to about 60 basepairs. To determine the expression level, a sample from the patient,optionally first subjected to a reverse transcription, is labelled andcontacted with the microarray in hybridization conditions, leading tothe formation of complexes between target nucleic acids that arecomplementary to probe sequences attached to the microarray surface. Thelabelled hybridized complexes are then detected and can be quantified orsemi-quantified. Labelling may be achieved by various methods, e.g. byusing radioactive or fluorescent labelling. Many variants of themicroarray hybridization technology are available to the man skilled inthe art (see e.g. the review by Hoheisel, Nature Reviews, Genetics,2006, 7:200-210).

In some embodiments, the nCounter® Analysis system is used to detectintrinsic gene expression. The basis of the nCounter® Analysis system isthe unique code assigned to each nucleic acid target to be assayed(International Patent Application Publication No. WO 08/124847, U.S.Pat. No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008. 26(3):317-325; the contents of which are each incorporated herein by referencein their entireties). The code is composed of an ordered series ofcolored fluorescent spots which create a unique barcode for each targetto be assayed. A pair of probes is designed for each DNA or RNA target,a biotinylated capture probe and a reporter probe carrying thefluorescent barcode. This system is also referred to, herein, as thenanoreporter code system. Specific reporter and capture probes aresynthesized for each target. The reporter probe can comprise at a leasta first label attachment region to which are attached one or more labelmonomers that emit light constituting a first signal; at least a secondlabel attachment region, which is non-over-lapping with the first labelattachment region, to which are attached one or more label monomers thatemit light constituting a second signal; and a first target-specificsequence. Preferably, each sequence specific reporter probe comprises atarget specific sequence capable of hybridizing to no more than one geneand optionally comprises at least three, or at least four labelattachment regions, said attachment regions comprising one or more labelmonomers that emit light, constituting at least a third signal, or atleast a fourth signal, respectively. The capture probe can comprise asecond target-specific sequence; and a first affinity tag. In someembodiments, the capture probe can also comprise one or more labelattachment regions. Preferably, the first target-specific sequence ofthe reporter probe and the second target-specific sequence of thecapture probe hybridize to different regions of the same gene to bedetected. Reporter and capture probes are all pooled into a singlehybridization mixture, the “probe library”. The relative abundance ofeach target is measured in a single multiplexed hybridization reaction.The method comprises contacting the sample with a probe library, suchthat the presence of the target in the sample creates a probepair-target complex. The complex is then purified. More specifically,the sample is combined with the probe library, and hybridization occursin solution. After hybridization, the tripartite hybridized complexes(probe pairs and target) are purified in a two-step procedure usingmagnetic beads linked to oligonucleotides complementary to universalsequences present on the capture and reporter probes. This dualpurification process allows the hybridization reaction to be driven tocompletion with a large excess of target-specific probes, as they areultimately removed, and, thus, do not interfere with binding and imagingof the sample. All post hybridization steps are handled robotically on acustom liquid-handling robot (Prep Station, NanoString Technologies).Purified reactions are typically deposited by the Prep Station intoindividual flow cells of a sample cartridge, bound to astreptavidin-coated surface via the capture probe, electrophoresed toelongate the reporter probes, and immobilized. After processing, thesample cartridge is transferred to a fully automated imaging and datacollection device (Digital Analyzer, NanoString Technologies). Theexpression level of a target is measured by imaging each sample andcounting the number of times the code for that target is detected. Foreach sample, typically 600 fields-of-view (FOV) are imaged (1376×1024pixels) representing approximately 10 mm2 of the binding surface.Typical imaging density is 100-1200 counted reporters per field of viewdepending on the degree of multiplexing, the amount of sample input, andoverall target abundance. Data is output in simple spreadsheet formatlisting the number of counts per target, per sample. This system can beused along with nanoreporters. Additional disclosure regardingnanoreporters can be found in International Publication No. WO 07/076129and WO07/076132, and US Patent Publication No. 2010/0015607 and2010/0261026, the contents of which are incorporated herein in theirentireties. Further, the term nucleic acid probes and nanoreporters caninclude the rationally designed (e.g. synthetic sequences) described inInternational Publication No. WO 2010/019826 and US Patent PublicationNo. 2010/0047924, incorporated herein by reference in its entirety.

Typically, the predetermined reference value is a threshold value or acut-off value. Typically, a “threshold value” or “cut-off value” can bedetermined experimentally, empirically, or theoretically. A thresholdvalue can also be arbitrarily selected based upon the existingexperimental and/or clinical conditions, as would be recognized by aperson of ordinary skilled in the art. For example, retrospectivemeasurement of expression levels in properly banked historical patientsamples may be used in establishing the predetermined reference value.The threshold value has to be determined in order to obtain the optimalsensitivity and specificity according to the function of the test andthe benefit/risk balance (clinical consequences of false positive andfalse negative). Typically, the optimal sensitivity and specificity (andso the threshold value) can be determined using a Receiver OperatingCharacteristic (ROC) curve based on experimental data. For example,after quantifying the expression level in a group of reference, one canuse algorithmic analysis for the statistic treatment of the determinedlevels in samples to be tested, and thus obtain a classificationstandard having significance for sample classification. The full name ofROC curve is Receiver Operator Characteristic Curve, which is also knownas receiver operation characteristic curve. It is mainly used forclinical biochemical diagnostic tests. ROC curve is a comprehensiveindicator that reflects the continuous variables of true positive rate(sensitivity) and false positive rate (1-specificity). It reveals therelationship between sensitivity and specificity with the imagecomposition method. A series of different cut-off values (thresholds orcritical values, boundary values between normal and abnormal results ofdiagnostic test) are set as continuous variables to calculate a seriesof sensitivity and specificity values. Then sensitivity is used as thevertical coordinate and specificity is used as the horizontal coordinateto draw a curve. The higher the area under the curve (AUC), the higherthe accuracy of diagnosis. On the ROC curve, the point closest to thefar upper left of the coordinate diagram is a critical point having bothhigh sensitivity and high specificity values. The AUC value of the ROCcurve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result getsbetter and better as AUC approaches 1. When AUC is between 0.5 and 0.7,the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy ismoderate. When AUC is higher than 0.9, the accuracy is quite high. Thisalgorithmic method is preferably done with a computer. Existing softwareor systems in the art may be used for the drawing of the ROC curve, suchas: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0,ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GBSTAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is determined bycarrying out a method comprising the steps of

a) providing a collection of samples from patients with decompensatedalcoholic cirrhosis;

b) providing, for each sample provided at step a), information relatingto the actual clinical outcome for the corresponding patient (i.e. theduration of the overall survival (OS));

c) providing a serial of arbitrary quantification values;

d) determining the expression level of the gene of interest for eachsample contained in the collection provided at step a);

e) classifying said samples in two groups for one specific arbitraryquantification value provided at step c), respectively: (i) a firstgroup comprising samples that exhibit a quantification value for levelthat is lower than the said arbitrary quantification value contained inthe said serial of quantification values; (ii) a second group comprisingsamples that exhibit a quantification value for said level that ishigher than the said arbitrary quantification value contained in thesaid serial of quantification values; whereby two groups of samples areobtained for the said specific quantification value, wherein the samplesof each group are separately enumerated;

f) calculating the statistical significance between (i) thequantification value obtained at step e) and (ii) the actual clinicaloutcome of the subjects from which samples contained in the first andsecond groups defined at step f) derive;

g) reiterating steps f) and g) until every arbitrary quantificationvalue provided at step d) is tested;

h) setting the said predetermined reference value as consisting of thearbitrary quantification value for which the highest statisticalsignificance (most significant) has been calculated at step g).

For example the expression level has been assessed for 100 samples of100 subjects. The 100 samples are ranked according to the expressionlevel. Sample 1 has the highest density and sample 100 has the lowestdensity. A first grouping provides two subsets: on one side sample Nr 1and on the other side the 99 other samples. The next grouping provideson one side samples 1 and 2 and on the other side the 98 remainingsamples etc., until the last grouping: on one side samples 1 to 99 andon the other side sample Nr 100. According to the information relatingto the actual clinical outcome for the corresponding patient, KaplanMeier curves are prepared for each of the 99 groups of two subsets. Alsofor each of the 99 groups, the p value between both subsets wascalculated. The predetermined reference value is then selected such asthe discrimination based on the criterion of the minimum p value is thestrongest. In other terms, the expression level corresponding to theboundary between both subsets for which the p value is minimum isconsidered as the predetermined reference value. It should be noted thatthe predetermined reference value is not necessarily the median value ofexpression levels. Thus in some embodiments, the predetermined referencevalue thus allows discrimination between a poor and a good prognosiswith respect to survival time for a patient. Practically, highstatistical significance values (e.g. low P values) are generallyobtained for a range of successive arbitrary quantification values, andnot only for a single arbitrary quantification value. Thus, in someembodiments, instead of using a definite predetermined reference value,a range of values is provided. Therefore, a minimal statisticalsignificance value (minimal threshold of significance, e.g. maximalthreshold P value) is arbitrarily set and a range of a plurality ofarbitrary quantification values for which the statistical significancevalue calculated at step g) is higher (more significant, e.g. lower Pvalue) are retained, so that a range of quantification values isprovided. This range of quantification values includes a “cut-off” valueas described above. For example, according to this specific embodimentof a “cut-off” value, the outcome can be determined by comparing theexpression level with the range of values which are identified. In someembodiments, a cut-off value thus consists of a range of quantificationvalues, e.g. centered on the quantification value for which the higheststatistical significance value is found (e.g. generally the minimum pvalue which is found). For example, on a hypothetical scale of 1 to 10,if the ideal cut-off value (the value with the highest statisticalsignificance) is 5, a suitable (exemplary) range may be from 4-6. Forexample, a patient may be assessed by comparing values obtained bydetermining the expression level of the gene of interest, where valuesgreater than 5 reveal a poor prognosis and values less than 5 reveal agood prognosis. In a another embodiment, a patient may be assessed bycomparing values obtained by measuring the expression level of the geneand comparing the values on a scale, where values above the range of 4-6indicate a poor prognosis and values below the range of 4-6 indicate agood prognosis, with values falling within the range of 4-6 indicatingan intermediate occurrence (or prognosis).

In some embodiments, when the expression levels of at least 2 genes aredetermined, a score which is a composite of said expression levels iscalculated and compared to its corresponding predetermined referencevalue, wherein when the score is higher than the predetermined referencevalue it is concluded that the patient will have a short survival timeand when the score is lower than the predetermined reference value, itis concluded that the patient will a long survival time. Typically thescore is the IFN score as described in the EXAMPLE.

The method of the present invention is particularly suitable fordetermining whether a patient with decompensated alcoholic cirrhosis iseligible to liver transplantation. Accordingly a further object relatesto a method of treating a patient with decompensated alcoholic cirrhosiscomprising determining the survival time of the patient by the method ofthe present invention and performing liver transplantation when it isconcluded that the patient will have a short survival time.

The invention will be further illustrated by the following FIGURES andexamples. However, these examples and FIGURES should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1. Forrest plot showing the prognostic value of 6 ISGs, our IFNscore, 2 cytokine genes and 3 well-known prognostic scores used inpatients with cirrhosis in univariate analysis. Ex vivo RT-qPCRexperiments were performed in PBMCs from 42-56 patients; however,results of relative mRNA expression were not obtained in someexperiments due to inefficient annealing of primers and patients whounderwent liver transplantation were counted as censored. The potentialrelation of cytokine gene levels to the risk of death (counted as event)was analyzed by Cox regression univariate analysis.

EXAMPLES Example 1

Patients and Methods

I—Patients

This protocol has been approved by the ethic committee (Comité deprotection des personnes Ile de France III) and informed consent wasobtained from every patients. 94 cirrhotic patients from the LiverDisease department of Beaujon Hospital (Assistance Publique—Hôpitaux deParis, Clichy, 92110, France) were selected according the prerequisitesof each experiment. As mentioned in the method section of part 1,patients enrolled in the study had severe decompensated biopsy-provenalcoholic cirrhosis. However, they were stable and did not haveuntreated or recently treated (less than 1 week) bacterial infection orgastrointestinal bleeding. Global characteristics of the patients aredisplayed in Table 1. Healthy subjects were selected among healthycaregivers. Fifteen to twenty milliliters of venous blood was sampled in3 mL EDTA tubes (Becton Dickinson, France) from patients and healthysubjects. Clinical and biological data were collected and relevantprognostic scores calculated.

TABLE 1 Characteristics of patients with decompensated alcoholiccirrhosis enrolled during the second part of this work. Variable n = 94Male 77 (81.9) Age (years) 57.5 (50-61) Child-Pugh Score 11 (9-12) MELDScore 19.3 (15.6-23.8) Serum Creatinine (μmol/L) 84.2 (57.0-97.0)Platelets (G/L) 103.0 (65.2-140) Leukocytes (G/L) 6.9 (5-9.4)Lymphocytes (G/L) 1.1 (0.8-1.7) Neutrophils (G/L) 4.4 (2.9-6.6)Monocytes (G/L) 0.8 (0.5-1) CRP 13 (6-29) Na (mmoL/L) 135 (131-137) CLIPSOFA score 6 (4-7) ACLF Grade 0 (0-0) Ascites 79 (84.0) Encephalopathy34 (36) Antibiotic 47 (50) B-blockers 39 (41.5) Corticosteroids 6 (6.6)Acute alcoholic hepatitis 24 (25.5) Results are expressed as number(percentage) or median (IQR)

II—PBMC and Monocyte Isolation and Culture, RNA Extraction, RT-qPCR

II-1. PBMC Isolation and Culture

PBMCs isolation and culture was performed as described in thecorresponding patients and methods section of the part 1 of this work.

PBMCs from patients and healthy subjects were left unstimulated orstimulated for four hours with 1 μg/mL LPS (TLR4 agonist, Escherichiacoli serotype 0111:B4, Sigma Aldrich St Louis, Mo., USA), 10 μg/mL PolyI:C (TLR3 agonist, Enzo Life Sciences, Farmingdale, N.Y., USA), 10 μg/mLpoly I:C+lipofectamin (RLR agonist, Life Technologies, Carlsbad, Calif.,USA) or 10 fold increasing concentrations of human IFN-β la (PBL AssayScience, Piscataway, N.J., USA) ranging from 1 UI/mL (5 ρg/mL) to 1000UI (5 μg/mL). In some experiments, PBMCs were stimulated with 1 μg/mLLPS (Escherichia coli serotype 0111:B4, Sigma Aldrich) for one houronly.

II-2. Monocyte Isolation and Culture

A reported corresponding patients and methods section of the part 1 ofthis work, after PBMC isolation, monocytes were separated from otherimmune cells using negative selection thanks to electromagnetic beads(Dynabeads®, Untouched human monocyte kit, Thermo Fischer scientific,Waltham, Mass., USA) following the manufacturer's protocol. Monocyteswere cultured according the protocols described for PBMCs.

II-3. Cycloheximide

PBMC from patients were isolated, counted and cultured according topreviously described protocol. Cells were preincubated or not for 15minutes with Cycloheximide (CHX, 10 μg/mL, Sigma, St Louis, Mo., USA), anatural inhibitor of protein synthesis and then left untreated orstimulated with 1 μg/mL LPS for 4 hours. At the end of culture time,cells were harvested and lysed as hereafter described.

II-4. Brefeldin A

PBMC from patients were isolated, counted and cultured according topreviously described protocol. Cells were incubated or not for 2 minuteswith Brefeldin A (BRF, Sigma, St Louis, Mo., USA) an inhibitor ofprotein secretion and then left untreated or stimulated with 1 μg/mL LPSfor 4 hours. At the end of culture time, cells were harvested and lysedas hereafter described.

II-5. IFN-α/β Receptor Chain 2 (IFNAR2) Blockade

PBMCs from patients and healthy subjects were isolated, counted andcultured according to previously described protocol with 1 μg/mL LPS. Asdescribed in previous studies [159], PBMCs were preincubated or not withwith anti-human IFN-α/β receptor chain 2 monoclonal antibody (30 μg/mL,PBL Assay Science, Piscataway, N.J., USA) or control isotopicimmunoglobulin fragment Fc (IgG2) and then left untreated or stimulatedwith 1 μg/mL LPS for 4 hours. At the end of the culture time, cells wereharvested and lysed as hereafter described.

II-6. LPS Tolerance Experiments

PBMCs from patients and healthy subjects were stimulated one or twotime(s) with LPS. They were stimulated or not a first time with 10 ng/mLLPS for 24 hours and then stimulated with 10 ng/mL LPS for 4 hours.Thus, on one hand, naïve PBMCs were only stimulated once with LPS for 4hours after a 24 hours-culture period without stimulation and, on theother hand, tolerant PBMCs were also stimulated with LPS for four hours,but after previous a 24 hour low dose LPS stimulation.

II-7. RNA Isolation

See the corresponding paragraph in the materiel and methods section ofthe part 1 of this manuscript.

II-8. Reverse Transcription

Genomic DNA was eliminated using DNase I, RNase free kit (Fermentas LifeSciences, USA) following the manufacturer's protocol. One microgram ofRNA was mixed with 1 μL of DNase and 1 μL of DNase I buffer in 10 μLvolume and incubated during 30 minutes at 37° C. in a PCR thermocycler(Applied Biosystems 2720, Singapore). DNase activity was interruptedadding 1 μL of EDTA (50 mM) and incubating the mix during 10 minutes at65° C. in the thermocycler. Reverse transcription was conducted usingThermo Fischer Scientific Verso cDNA Synthesis kit following themanufacturer's protocol (Thermo Fischer scientific, Waltham, Mass.,USA). The previously obtained mix was completed with oligonucleotidesoligo-DT primers, deoxynucleotides, buffer, enhancer and VERSO enzyme.The mix was incubated during 60 minutes at 42° C. Reverse transcriptaseenzyme activity was stopped by warming at 95° C. and cDNA were stored à−20° C.

II-9. Real Time Quantitative PCR Analysis (RT-qPCR)

Quantitative PCR analysis was used to assess mRNA expression in immunecells. Specific Primers for studied genes were designed using Primer3webversion 4.0. Percent PCR amplification efficiencies (E) were calculatedfor each primer as E=(10−1/slope−1)×100, using the slope of the semi-logregression plot of Ct versus log input of cDNA (10-fold dilution seriesof five points). A threshold of 5% above or below 100% efficiency wasapplied (i.e. an efficiency between 1.9 and 2.1).[150] Then DNAsolutions were diluted from 1/15 to 1/30 according the mRNA expressionlevel. Ninety six well plates were used. Each wells was filled with 5 μLof diluted cDNA solution and a mixed solution containing 10 μL of SYBRGreen I enzyme (ABgene, Thermo Scientific, Waltham, USA), 3 μL ofdistilled water and 2 μL of specific primers. Plates were sealed withplastic films and inserted in a Light Cycler 480 (Roche DiagnosticsGmbH, Mannheim, Germany). Forty cycle thermocycling sequences wereconducted. A melting curve was systematically run at the end thereaction to verify that the used primer pair produced a single product.Cycle Threshold (Ct) level was obtained for each gene tested. Allanalysis were conducted in duplicate. To favor technical reproducibilityof the qPCR experiments, sample with standard deviation of the crossingpoint >0.3 from the mean Ct were excluded from analysis. Results wereexpressed as the N-fold differences in target gene expression relativeto the GAPDH housekeeping gene expression in unstimulated PBMCs fromhealthy subjects. Results were determined as 2^(−ΔCtsample) where theΔCt value of the sample was determined by substracting the average Ctvalue of the target gene from the average Ct value of the GAPDH gene. Asdescribed elsewhere, the obtained values of the samples were thennormalized such that the median of the corresponding gene values inPBMCs from healthy subjects was 1.[160]

As previously described, the median fold change of 8 ISGs, when comparedwith the median of the combined healthy controls, was used to create aninterferon score for each patient (Yao, Y., et al., Development ofPotential Pharmacodynamic and Diagnostic Markers for Anti-IFN-alphaMonoclonal Antibody Trials in Systemic Lupus Erythematosus. Hum GenomicsProteomics, 2009. 2009.; Rice, G. I., et al., Assessment ofinterferon-related biomarkers in Aicardi-Goutieres syndrome associatedwith mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR:a case-control study. Lancet Neurol, 2013. 12(12): p. 1159-69.).Selected genes were the 7 most highly expressed interferon stimulatedgenes in LPS-stimulated «healthy» cells (i.e. IFIT1, CXCL10, MX2, IFIH1,DDX58, OAS2 and TRIM22) and GBP4 (as member of the physiopathologicallyimportant GBP family).

III—Statistical Analysis

Quantitative variables were when expressed as median (Interquartilerange) and categorical variables as number (percentage). Comparisonsbetween groups of quantitative variables were performed usingMann-Whitney test and Wilcoxon test appropriate. All tests weretwo-sided and used a significance level of 0.05. Data handling andanalysis were performed with SPSS 22.0 (SPSS Inc., Chicago, Ill.) andGraphPad Prism version 6 for Mac OS X. Cumulative survival wascalculated by Kaplan-Meier method. Patients who underwent livertransplantation were counted as censored. The potential relation ofcytokine gene levels with the risk of death was analyzed by Coxregression univariate analysis. Each variable achieving a p value <0.05was introduced into a Cox model with MELD score in order to determinewhether these variables had a prognostic value independently of MELDscore, as previously reported.

Results

I—LPS-Induced Expression of Genes Identified as ISGs by InterferomeDatabase is Defective in PBMCs from Patients with DecompensatedAlcoholic Cirrhosis

Our expression microarrays revealed a core of 444 genes upregulated in“healthy” PBMCs but either non-regulated or downregulated in cirrhoticPBMCs belonging to the “defense response to virus” GO BP. Interestingly,quiering the interferome public database(interferome.its.monash.edu.au), we found that 174 of these genes wereclassified as ISGs among which, 168 were type 1 IFN-stimulated genes.Moreover, five other genes (IDO1, LAG3, OAS3, RSAD2 and USP18)classified as ISGs by interferome were induced by LPS both in “healthy”and cirrhotic PBMCs but their expression was more than two fold higherin “healthy” than in cirrhotic cells. Finally, one gene was downregulated by LPS in “cirrhotic” PBMCs whereas it was upregulated by LPSin “healthy PBMCs”.

Next, we selected a set of 46 type-1 ISGs according to their functionsand we measured, using RT-qPCR, the relative steady-state levels of mRNAfrom each gene in PBMCs stimulated with LPS, from an independent cohortof 33 patients with decompensated alcoholic cirrhosis and 17 healthysubjects. As a control, we also measured in PBMCs from the same patientsunder the same conditions the expression of 6 cytokine genes (IL10,IL12B, IL6, IL1B, IL8, and TNF) and 4 chemokine genes (CXCL1, CXCL2,CXCL3, CXCL5).

LPS induced all the 46 genes in both groups. However, the LPS-inducedlevel of expression of 70% (32 over the 46 studied genes) of ISGs wassignificantly lower in “cirrhotic” PBMCs than in “healthy” PBMCs. Usingwestern blotting, we confirmed this defect at the protein level. Indeed,we showed a lower expression of OAS2 (encoded by the ISG OAS2) proteinlevels in LPS-stimulated cirrhotic PBMCs, as compared to LPS-stimulated“healthy PBMCs.

Taken together, our results show, in PBMCs from patients withdecompensated alcoholic cirrhosis, a defective LPS-induced expression ofa large group of genes identified as type 1 IFN ISGs by interferome.This defective induction of ISGs in cirrhotic PBMCs was not due to anincreased cellular death. Indeed, we quantified cell lysis and celldeath by measuring lactate deshydrogenase (LDH) in supernatant from 4h-LPS stimulated healthy (n=14) and cirrhotic (n=14) PBMCs using acolorimetric assay. We found a very low mean percentage of cytotoxicitythat did not differ between healthy and cirrhotic cells (0.6% vs 1.1%,p=0.51). Furthermore, we found several cytokine and chemokine genes suchTNF (encoding TNF-α), CXCL2, CXCL3 and CXCL5 (encoding ENA-78) withsignificantly higher LPS-induced expression in cirrhotic PBMCs. Finally,we found the similar defect of ISG expression after a one hour LPSstimulation.

II—Verification of the True ISG Status of the Genes

Functions of each gene were analyzed and, for practical reasons, a“restricted” set of 21 genes of interest identified as ISGs byinterferome database and whose expression was altered in LPS-stimulatedcirrhotic PBMCs was selected for experiments performed thereafter. Weverified the “bona-fide” ISG status of each gene of interest using twoapproaches. First, we wondered if type I-IFN receptor (IFNAR) blockingwould result in an inhibition of the induction of these genes by LPS.Thus, PBMCs from five patients with decompensated alcoholic cirrhosiswere preincubated, or not, with a type I-IFN-α/β receptor chain 2(IFNAR2) antagonist (30 μg/mL) for 15 minutes before being stimulatedwith LPS. We found that preincubation of cirrhotic cells with IFNAR2antagonist abolished LPS-induced expression of 19 genes (95%), whereasincubation with the isotype control antibody did not. This inhibitoryeffect was specific for ISG as far as LPS-induced expression of genesencoding cytokines not known to belong to ISGs was not altered bypreincubation with IFNAR2. Furthermore, IFNAR antibody had the sameeffect on ISG induction in LPS stimulated “healthy” PBMCs.

Secondly, we wondered if an IFN-β-stimulation would result in aninduction of these genes. Thus, PBMCs from eight patients withdecompensated alcoholic cirrhosis were stimulated with 10 UI/mL IFN-β.18 of the 19 genes whose induction by LPS was sensitive to IFNARantagonist were also induced by a 10 UI/mL-IFN-β stimulation. Thus,altogether, we found that among our restricted set of 21 genes ofinterest identified as ISGs by interferome public database, 18 (86%)were “bona-fide” ISGs in PBMCs from patients with decompensatedalcoholic cirrhosis.

III—Construction of an ISG Signature Named IFN Score

As previously performed (Yao, Y., et al., Development of PotentialPharmacodynamic and Diagnostic Markers for Anti-IFN-alpha MonoclonalAntibody Trials in Systemic Lupus Erythematosus. Hum GenomicsProteomics, 2009. 2009.; Rice, G. I., et al., Assessment ofinterferon-related biomarkers in Aicardi-Goutieres syndrome associatedwith mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR:a case-control study. Lancet Neurol, 2013. 12(12): p. 1159-69.) wedeveloped an ISG signature that was named IFN score to quantify themagnitude of the global downregulation of ISGs in PBMCs from patientswith cirrhosis. Briefly, the IFN score was calculated for each patientand healthy subject as the median fold change of 8 ISGs normalized withthe median of the combined healthy controls. The 7 most highly expressedISGs (among the 18 bona fide ISGs identified above) in LPS-stimulated“healthy” cells (i.e. IFIT1, CXCL10, MX2, IFIH1, DDX58, OAS2 and TRIM22)were used in IFN score. In order to include a member of thephysiopathologically important GBP family, we also selected GBP4, themost highly LPS-induced member of GBP family. Thus 8 ISGs were includedin the IFN score. Their name, corresponding locus, encoded protein andfunctions are displayed in Table A.

Calculation the IFN score in LPS-stimulated PBMCs from previouslymentioned decompensated cirrhotic patients and healthy subjects allowedus to confirmed the global defective induction of ISGs in immune cellsfrom patients. It is important to note that IFN score values did notcorrelate with leukocyte, monocyte or lymphocyte counts and antibioticor beta-blocker treatment of patients.

IV—Defects in Basal and LPS-Induced ISG Expression are Linked to theSeverity of Cirrhosis

As in patients with cirrhosis, alterations of innate immune response andbacterial translocation are closely linked to the severity of cirrhosis,we investigated unstimulated and LPS stimulated ISG expression in PBMCsfrom patients with increasing severity of cirrhosis as reflected byChild-Pugh score. 8 patients with Child-Pugh grade A, 20 with Child-Pughgrade B and 45 with Child-Pugh grade C cirrhosis were enrolled (Table2).

TABLE 2 Characteristics of enrolled Child-Pugh A, Child-Pugh B, andChild-Pugh C patients. Child Pugh A Child Pugh B Child-Pugh C Number ofpatients, n 7 20 28 Sex (M/F) 6 (0.85) 17 (0.8) 23 (82) Age, yr 59.5(53-66) 57 (51-60) 59 (53-63) Child-Pugh Score 5 (5-5.2) 8.5 (8-9) 12(11-13) MELD 8.9 (8-10) 13 (10-16) 21.2 (18-27) Serum creatinine 68.5(66-71) 74.5 (67-97) 67 (57-88) (μmol/L) Platelets (G/L) 124 (81-147)113 (85-155) 96 (56-120) Leukocytes (G/L) 5.2 (3.5-5.8) 6 (4.4-7.7) 7.3(4.8-10.1) Lymphocytes (G/L) 1.16 (0.8-1.6) 1.07 (0.8-1.5) 1.03(0.7-1.6) Monocytes (G/L) 0.39 (0.3-0.5) 0.69 (0.5-0.9) 0.78 (0.5-1.1)CRP (mg/L) 2.5 (2-4) 11 (5-15) 16 (6-30) Na (mmol/L) 138 (135-138) 135(131-137) 134 (130-137) CLIP SOFA score 1.5 (1-2) 3 (2.7-4) 7 (5-8) ACLFGrade 0 (0) 0 (0-0) 0 (0-1) Ascites, n (%) 0 (0) 15 (75) 24 (87)Encephalopathy 0 (0) 2 (10) 13 (45) Antibiotics, n (%) 0 (0) 5 (25) 15(55) β-blocker, n (%) 2 (28) 5 (25) 10 (37) Corticosteroids, n (%) 0 (0)0 (0) 3 (11) Histological alcoholic 0 (0) 2 (10) 9 (33) hepatitis, n (%)

As shown by lower IFN score values, we found that the defect in ISGexpression of both unstimulated and LPS-stimulated immune cellssignificantly worsen with the severity of cirrhosis. Whereas, IFN scoredid not statistically differ between Child A patients and healthysubjects, it was significantly lower in Child-Pugh B and Child-Pugh Cpatients as compared to Child A ones. Thus, the lower ISG response seemsto be restricted to the more severe cirrhotic patients.

V—Impact of ISG Expression on Patient Prognosis

Finally, we aimed at demonstrating the paramount importance of the ISGsystem and its regulation in patients with cirrhosis by investigatingthe impact of its lower activation on patient outcome, we prospectivelymonitored survival of patients in whom PBMC ISG expression was measured.40 patients (40%) died during a median (IQR) follow-up of 6.55(1.32-12.60) months. Using cox model, we found that, in unstimulatedcells, the value of our IFN score and the expression of six out of the18 true ISGs of interest were associated with patient outcome inunivariate analysis (FIG. 1). Interestingly, the lower the IFN scorevalue and the ISG expression were, the better the outcome. Thesefindings are consistent with the hypothesis of a beneficialdownregulation of ISG expression in patients with severe cirrhosis toavoid immunopathology. The major importance of the ISG system wasemphasized by the fact that the odds ratio of IFN score value inunstimulated cells for predicting death was much more higher than thoseof usual cirrhotic patients prognostic scores such as MELD score,Child-Pugh score, CLIF-SOFA score and ACLF grade (FIG. 1). Furthermore,unlike ISGs, unstimulated expression of usual cytokine gene did not haveany prognostic value (FIG. 1). Expression of ISGs and cytokine genesafter LPS stimulation were not associated with outcome. In multivariateanalysis, high basal IFN score value remained a risk factor of deathindependently of MELD score, CLIF SOFA score and CRP level (Table 3).Interestingly, half of these deaths were due to septic shock and 86% dueto multiple organ failure (Table 4). At the gene level, high basal MX2and OAS2 expressions also predicted death independently of MELD scoreand CLIF SOFA. Interestingly, in our cohort IFN score had the best deathpredicting ability for patients with MELD score lower than 19, themedian MELD score of our cohort. Thus, our IFN score may allow an earlydetection of patients with stable decompensated cirrhosis whoparticularly are at risk of death in the next future. Indeed, thissubgroup of patients may derive the maximum benefit from livertransplantation.

TABLE 3 Prognostic value of the basal IFN score, MX2 and OAS2expressions and of usual cirrhotic patient prognostic scores (MELD scoreand CLIF SOFA score). Ex vivo RT-qPCR experiments were performed inPBMCs from 42-56 patients. However, results of relative mRNA expressionwere not obtained in some experiments due to inefficient annealing ofprimers and patients who underwent liver transplantation were counted ascensored. For bivariate analyses: we fitted 5 Cox models including MELDscore with each variable achieving a p < 0.05 successively, in order todetermine if these variables had a prognostic value independently ofusual cirrhotic patient prognostic scores (MELD score, CLIF SOFA scoreand CRP levels). 95.0% CI Number of Total number for risk ratio eventsof patients Risk ratio Lower Upper P value First model 14 43 IFN Scorebasal 3.583 1.177 10.905 .025 MELD score 1.071 .993 1.155 .074 Secondmodel 14 43 IFN Score basal 3.958 1.358 11.533 .012 CLIF SOFA score1.126 .904 1.403 .288 Third model 13 42 IFN Score basal 4.072 .007 CRP1.007 .454 Fourth model 21 56 MX2 basal 1.346 1.077 1.681 .009expression MELD score 1.091 1.020 1.167 .011 Fifth model 21 56 MX2 basal1.416 1.144 1.752 .001 expression CLIF SOFA score 1.301 1.063 1.591 .010Sixth model 20 55 OAS2 basal 2.492 1.053 5.901 .038 expression MELDscore 1.060 .984 1.141 .122 Seventh model 20 55 OAS2 basal 2.754 1.2356.140 .013 expression CLIF SOFA score 1.153 .930 1.429 .195

TABLE 4 Causes of death (n = 14) in the cohort of patients in whom IFNscore was measured (n = 43) Cause of death n (%) Septic shock 7 (50) MOFwithout aetiology 3 (22) MOF following hemorrhagic shock 2 (14)Hepatorenal syndrome 1 (7)  Unknown 1 (7) 

Example 2

Baseline ISG Expression is Related to Outcome in Cirrhosis

The inventors prospectively followed-up a cohort of patients aftermeasurement of PBMC ISG expression at enrollment. Forty-one patientsdied during a median follow-up of 6.55 (1.32-12.60) months. A univariateCox model showed that higher mortality was significantly associated witha higher model of end-stage liver disease (MELD) score, a higher IFNscore in unstimulated cells, and higher expressions of DDX58, MX2, OAS2,DDX60, IFI44, IFIT1, IFIT5 and IRF1 in unstimulated cells (FIG. 1; Table5). Unlike ISGs, cytokine gene expression (including the ISG CXCL10) didnot significantly predict death in unstimulated cells (FIG. 1; Table 5).Only one (IFI35) of the genes measured in LPS-stimulated cellssignificantly predicted death (Table 5). Bivariate analysis includingthe baseline IFN and MELD scores only showed the former to besignificantly predictive of death (Table 6). These results are mainlyexplained by the intrinsic prognostic value of higher baseline MX2,OAS2, and DDX58. Finally, higher baseline expressions of IFI44 and DDX60were significant predictors of death, independent of the MELD score(Table 6).

TABLE 5 Prognostic value of the MELD score and ex vivo ISG and cytokinegene expression in unstimulated and LPS-stimulated PBMCs from patientswith cirrhosis, Related to FIG. 1. Genes highlighted had significantprognostic value (p < 0.05). Total Variables in the number of Number ofRisk 95.0% CI for Equation patients events ratio risk ratio p valuePrognostic scores MELD score 98 41 1.120 1.065 1.177 <0.001 Geneexpression in unstimulated cells Interferon-stimulated genes AIM2 15 8.965 .162 5.746 .969 BST2 18 10 1.199 .311 4.623 .792 CXCL10 60 20 1.045.959 1.139 .316 CXCL11 13 8 1.285 .272 6.065 .751 DDX58 55 21 1.7171.191 2.476 .004 DDX60 46 16 2.526 1.391 4.586 .002 DHX58 49 18 1.209.729 2.005 .461 EIF2AK2 18 9 1.128 .690 1.845 .631 GBP1 17 9 1.194 .5212.738 .676 GBP2 16 9 .468 .087 2.526 .378 GBP3 47 17 1.330 .880 2.011.176 GBP4 53 19 1.217 .723 2.047 .459 GBP5 16 9 .367 .095 1.416 .146HERC5 18 10 1.448 .707 2.966 .312 IDO1 17 9 1.073 .957 1.203 .228 IFI1616 8 .527 .054 5.099 .580 IFI35 54 21 1.067 .905 1.259 .440 IFI44 17 91.711 1.091 2.683 .019 IFIH1 54 20 1.551 .982 2.449 .060 IFIT1 50 181.168 1.032 1.322 .014 IFIT2 16 9 1.339 .964 1.862 .082 IFIT3 49 181.016 .987 1.047 .282 IFIT5 40 14 1.293 1.008 1.659 .043 IFITM3 18 101.084 .903 1.300 .387 IFNB1 37 17 1.157 .907 1.476 .241 IFNG 41 13 .870.584 1.295 .493 IRF1 56 22 1.699 1.049 2.752 .031 ISG20 17 9 .624 .2001.950 .418 MB21D1 48 20 1.611 .761 3.411 .213 MCOLN2 15 8 1.002 .4462.251 .996 MOV10 54 19 1.768 .867 3.605 .117 MX1 15 10 1.176 .767 1.801.457 MX2 56 21 1.416 1.151 1.741 .001 OAS1 16 9 1.914 .799 4.585 .145OAS2 55 20 3.359 1.634 6.908 .001 PNPT1 17 9 1.048 .262 4.199 .947 RSAD217 10 1.108 .920 1.335 .280 SMCHD1 18 10 .904 .495 1.651 .742 TLR3 49 191.341 .578 3.115 .495 TLR7 52 20 1.449 .479 4.384 .511 TREX1 50 19 .968.597 1.570 .895 TRIM14 55 21 1.930 .982 3.793 .057 TRIM21 47 15 2.156.775 5.995 .141 TRIM22 48 18 1.180 .579 2.403 .649 TRIM25 16 9 .704 .1742.848 .623 TRIM5 16 9 1.193 .772 1.843 .426 USP18 13 8 .901 .407 1.995.798 XRN1 18 10 1.211 .158 9.249 .854 ZBP1 52 19 1.548 .937 2.558 .088Cytokine genes IL10 81 34 1.152 .643 2.063 .634 IL6 52 20 .989 .9661.012 .353 IL1B 70 28 1.020 .978 1.063 .355 IL8 47 22 .999 .898 1.111.980 CXCL1 50 22 1.014 .967 1.063 .579 CXCL2 52 23 .989 .925 1.057 .746CXCL3 51 22 .977 .931 1.025 .337 CXCL5 48 21 .955 .889 1.027 .214 TNF 3717 .362 .103 1.275 .114 Gene expression in LPS-stimulated cellsInterferon-stimulated genes AIM2 7 6 1.562 .616 3.959 .348 BST2 10 71.823 .551 6.031 .325 CXCL10 40 12 .991 .945 1.039 .704 CXCL11 8 5 .993.811 1.216 .948 DDX58 33 12 1.004 .958 1.052 .883 DDX60 28 10 1.030 .8661.225 .739 DHX58 31 11 .970 .796 1.182 .763 EIF2AK2 15 9 .779 .581 1.046.097 GBP1 10 7 1.047 .902 1.215 .546 GBP2 10 7 1.402 .734 2.677 .307GBP3 28 11 .993 .874 1.128 .915 GBP4 33 12 1.009 .891 1.141 .891 GBP5 107 1.115 .913 1.363 .286 HERC5 10 7 1.019 .781 1.329 .891 IDO1 10 7 .989.944 1.036 .650 IFI16 17 9 1.386 .615 3.126 .431 IFI35 26 10 1.313 1.0071.711 .044 IFI44 19 10 .994 .937 1.053 .830 IFIH1 33 12 1.005 .958 1.055.827 IFIT1 31 12 1.021 .950 1.097 .573 IFIT2 9 6 1.033 .847 1.261 .748IFIT3 31 10 1.058 .930 1.203 .392 IFIT5 19 7 1.149 .648 2.038 .635IFITM3 9 6 1.032 .781 1.364 .825 IFNB1 23 8 .991 .920 1.068 .812 IFNG 288 1.001 .998 1.003 .716 IRF1 32 12 1.186 .851 1.652 .315 ISG20 10 71.059 .724 1.550 .766 MB21D1 31 12 1.068 .689 1.658 .768 MCOLN2 10 71.193 .937 1.520 .153 MOV10 33 12 1.148 .936 1.408 .186 MX1 10 7 1.062.832 1.354 .631 MX2 33 12 1.046 .978 1.118 .189 OAS1 9 6 1.249 .7612.050 .379 OAS2 33 12 1.075 .941 1.228 .289 PNPT1 19 10 1.529 .490 4.764.464 RSAD2 9 6 .977 .803 1.188 .812 SMCHD1 9 6 1.632 .780 3.413 .193TLR3 26 10 .782 .457 1.338 .370 TLR7 31 12 .962 .591 1.567 .876 TREX1 2810 1.078 .754 1.541 .680 TRIM14 32 12 1.079 .788 1.477 .637 TRIM21 31 101.324 .913 1.918 .139 TRIM22 33 12 .989 .854 1.145 .881 TRIM25 8 6 .688.273 1.735 .428 TRIM5 10 7 1.256 .688 2.294 .458 USP18 10 7 1.173 .8181.681 .386 XRN1 10 7 1.241 .449 3.425 .677 ZBP1 31 12 1.091 .984 1.210.098 Cytokine genes IL10 56 24 .982 .939 1.026 .414 IL6 50 19 1.0001.000 1.000 .968 IL1B 54 21 .996 .984 1.008 .491 IL8 50 23 1.002 .9971.007 .440 CXCL1 46 21 1.002 .998 1.005 .360 CXCL2 45 20 1.000 .9991.002 .539 CXCL3 48 22 1.001 .998 1.005 .425 CXCL5 46 22 .987 .966 1.009.244 TNF 41 20 1.002 .993 1.010 .738

TABLE 6 Baseline ISG expression is related to patients' outcome. Resultsof analysis using a bivariate model. 95.0% CI Number Total number forrisk ratio of events of patients Risk ratio Lower Upper P value Firstmodel 14 43 IFN Score basal 3.583 1.177 10.905 .025 MELD score 1.071.993 1.155 .074 Second model 21 56 MX2 basal 1.346 1.077 1.681 .009expression MELD score 1.091 1.020 1.167 .011 Third model 20 55 OAS2basal 2.492 1.053 5.901 .038 expression MELD score 1.060 .984 1.141 .122Fourth model 10 26 IFI35 basal 1.393 1.043 1.861 .025 expression MELDscore 1.178 1.026 1.351 .020 Fifth model 9 17 IFI44 basal 1.635 1.0172.627 .042 expression MELD score 1.032 .936 1.136 .529

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1. A method for predicting the survival time of a patient withdecompensated alcoholic cirrhosis comprising i) determining theexpression level of OAS2 or MX2 in a sample of peripheral bloodmononuclear cells obtained from the patient, ii) comparing the leveldetermined at step i) with a predetermined reference value and iii) andconcluding that the patient will have a short survival time when thelevel determined at step i) is higher than its predetermined referencevalue or concluding that the patient will have a long survival time whenthe level determined at step i) is lower than the predeterminedreference value.
 2. The method of claim 1 wherein the expression levelsof OAS2 and MX2 are determined at step i) and compared to theircorresponding predetermined reference value.
 3. The method of claim 1wherein the expression level of at least one further gene is determinedand compared to its corresponding predetermined value.
 4. The method ofclaim 1 wherein the gene for which the expression is further determinedis selected from the group consisting of IFIT1, CXCL10, IFIH1, DDX58,TRIM22 and GBP4.
 5. The method of claim 4 wherein the expression levelsof 2, 3, 4, 5, 6, 7 or 8 genes are determined.
 6. The method of claim 1wherein the expression levels of OAS2, MX2, IFIT1, CXCL10, IFIH1, DDX58,TRIM22 and GBP4 are determined in the sample and compared to theircorresponding predetermined reference values.
 7. The method of claim 1wherein the expression level of the gene is determined by RT-PCR.
 8. Themethod of claim 1 wherein when the expression levels of at least 2 genesare determined, a score which is a composite of said expression levelsis calculated and compared to its corresponding predetermined referencevalue, wherein when the score is higher than the predetermined referencevalue it is concluded that the patient will have a short survival timeand when the score is lower than the predetermined reference value, itis concluded that the patient will a long survival time.
 9. A method oftreating a patient with decompensated alcoholic cirrhosis comprisingdetermining the survival time of the patient by the method of claim 1and performing liver transplantation when it is concluded that thepatient will have a short survival time.